Retinoic acid (RA) is known to have an important signalling role in the regulation of embryonic development and cell differentiation. The biological effects of RA are transduced via two classes of nuclear receptors, designated retinoic acid receptors (RARα, RARβ and RARγ) and retinoid X receptors (RXRα, RXRβ and RXRγ) (Giguere et al., (1987), Nature, 350, 624-629).
Many studies have been made of the role of RA and its receptors in the process of chondrogenesis and skeletal development. For example, down regulation of RARα has been stated to be important for chondrogenic expression (Cash et al. (1997) J. Cell Biol. 136, 445-457). Down regulation of RAR-β2 using antisense oligonucleotides was found to stimulate chondrogenesis and thus it was suggested that RAR-β2 helps to prevent mesenchymal cells from expressing their chondrogenic bias. Underhill et al. (1998) Micro. Res. Tech. 43, 137-155 reported that abnormal expression of RARs inhibits chondrogenesis. However, it was also found that absence of RAR can lead to deficiencies in cartilage formation while also promoting chondrogenesis at ectopic sites.
Addition of RA and RAR-specific agonists has been shown to inhibit cartilage formation in limb bud micromass cultures, and act as a teratogen in vivo to negatively affect skeletal development (Kistler (1987) Arch. Toxicol. 60, 403-414; Kochhar (1973) Teratology 7, 289-295; Kochhar and Aydelotte (1974) J. Embryol. Exp. Morph. 31, 721-734; Kwasigrich and Kochhar (1980) Anat. Embryol. 161, 105-113. In contrast, addition of retinoic acid to micromass cultures has also been demonstrated to stimulate cartilage formation (Paulsen et al. (1994) Dev. Dynam. 201, 310-323; Paulsen et al. (1994) Dev. Biol. 30A, 181-186).
Addition of an RAR antagonist completely reversed the inhibitory action of an RAR agonist on chondrocyte differentiation in rat and mouse embryo limb bud mesenchymal cells in vitro (Eckhardt and Schmitt (1994) Toxicol. Letters, 70, 299-308; Kocchar et al. (1998) Int. J. Dev. Biol., 42, 601-608). These authors, however, found that the antagonist alone had no effect on limb bud mesenchymal cell differentiation.
The precise role of RARs in chondrogenesis was unclear from these studies. Specifically, it was unclear whether RARs functioned to inhibit chondrogenesis or stimulate that process. Overall, the role of RAR antagonists in the process of chondrogenesis was not clearly established.